Figure 8. Characterization of tepsin, a... | Rockefeller University Press

Figure 8.

$Figure 8. Characterization of tepsin, a new AP-4–associated protein. (A) Schematic diagrams of tepsin from organisms belonging to all five eukaryotic supergroups. All tepsins have an N-terminal ENTH domain and a central VHS/ENTH-like domain. (B) Western blots of whole cell lysates and coated vesicle (CV) fractions from primary human fibroblasts. Tepsin has two isoforms. Although the total amount of tepsin is unchanged in cells with mutations in the AP-4 β and μ subunits (AP4B1* and AP4M1*, respectively), the tepsin no longer co-fractionates with coated vesicles. (C) Immunofluorescence double labeling for tepsin and the AP-4 ε subunit in a primary cortical rat neuron. The two proteins show extensive colocalization. Bar, 10 µm. (D) Immunofluorescence double labeling for tepsin and AP-4 ε in primary human fibroblasts. Although the two proteins colocalize in a punctate perinuclear pattern in control cells, this pattern is absent in AP-4–deficient cells. Bar, 10 µm. (E) Western blots of immunoprecipitates of the AP-4 β subunit from extracts of control and AP-4–deficient fibroblasts, probed for AP-4 subunits and tepsin. Tepsin coimmunoprecipitates with AP-4 only in the control extracts. (F) PNS and coated vesicle fraction from control, AP-4–depleted, and tepsin-depleted HeLa cells. Although tepsin requires AP-4 for incorporation into coated vesicles, AP-4 can still be incorporated in the absence of tepsin. (G) GST pull-downs from tepsin-GFP–expressing HeLa cells. Tagged tepsin specifically interacts with the AP-4 β “ear” domain. (H) GST pull-downs from HeLa cells were analyzed by mass spectrometry. Peptides, unique matched peptides; count, total number of matched peptides. Tepsin is only pulled down with the AP-4 β ear. Positions of protein molecular mass markers are indicated in kilodaltons in B and E–G. In some cases, the apparent protein molecular weight is shown instead (indicated by ∼); this was estimated from the average migration of the protein relative to molecular weight markers on at least three gels.$

Characterization of tepsin, a new AP-4–associated protein. (A) Schematic diagrams of tepsin from organisms belonging to all five eukaryotic supergroups. All tepsins have an N-terminal ENTH domain and a central VHS/ENTH-like domain. (B) Western blots of whole cell lysates and coated vesicle (CV) fractions from primary human fibroblasts. Tepsin has two isoforms. Although the total amount of tepsin is unchanged in cells with mutations in the AP-4 β and μ subunits (AP4B1* and AP4M1*, respectively), the tepsin no longer co-fractionates with coated vesicles. (C) Immunofluorescence double labeling for tepsin and the AP-4 ε subunit in a primary cortical rat neuron. The two proteins show extensive colocalization. Bar, 10 µm. (D) Immunofluorescence double labeling for tepsin and AP-4 ε in primary human fibroblasts. Although the two proteins colocalize in a punctate perinuclear pattern in control cells, this pattern is absent in AP-4–deficient cells. Bar, 10 µm. (E) Western blots of immunoprecipitates of the AP-4 β subunit from extracts of control and AP-4–deficient fibroblasts, probed for AP-4 subunits and tepsin. Tepsin coimmunoprecipitates with AP-4 only in the control extracts. (F) PNS and coated vesicle fraction from control, AP-4–depleted, and tepsin-depleted HeLa cells. Although tepsin requires AP-4 for incorporation into coated vesicles, AP-4 can still be incorporated in the absence of tepsin. (G) GST pull-downs from tepsin-GFP–expressing HeLa cells. Tagged tepsin specifically interacts with the AP-4 β “ear” domain. (H) GST pull-downs from HeLa cells were analyzed by mass spectrometry. Peptides, unique matched peptides; count, total number of matched peptides. Tepsin is only pulled down with the AP-4 β ear. Positions of protein molecular mass markers are indicated in kilodaltons in B and E–G. In some cases, the apparent protein molecular weight is shown instead (indicated by ∼); this was estimated from the average migration of the protein relative to molecular weight markers on at least three gels.